1. Field of the Invention
The present invention concerns nucleic acid sequences which code for carcinoembryonic antigen (CEA) antigen family peptide sequences.
2. Background Information
Carcinoembryonic antigen was first described by Gold and Freedman, J. Exp. Med., 121, 439-462, (1965). CEA is characterized as a glycoprotein of approximately 200,000 molecular weight with 50-60% by weight of carbohydrate. CEA is present during normal human fetal development, but only in very low concentration in the normal adult intestinal tract. It is produced and secreted by a number of different tumors.
CEA is a clinically useful tumor marker for the management of colorectal cancer patients. CEA can be measured using sensitive immunoassay methods. When presurgical serum levels of CEA are elevated, a postsurgical drop in serum CEA to the normal range typically indicates successful resection of the tumor. Postsurgical CEA levels that do not return to normal often indicate incomplete resection of the tumor or the presence of additional tumor sites in the patient. After returning to normal levels, subsequent rapid rises in serum CEA levels usually indicate the presence of metastages. Slower postsurgical rises from the normal level are most often interpreted to indicate the presence of new primary tumors not previously detected. Post surgical management of colon cancer patients is thus facilitated by the measurement of CEA.
CEA is a member of an antigen family. Because of this, the immunoassay of CEA by presently available methods is complicated by the fact that CEA is but one of several potentially reactive antigens. There have been at least sixteen CEA-like antigens described in the literature. Since some of these appear to be the same antigen described by different investigators, the actual number of different antigens is somewhat less than this number. Nonetheless, there is a complex array of cross-reactive antigens which can potentially interfere with an immunoassay of the CEA released by tumors. It is known that serum levels of CEA-like antigens are elevated in many non-cancerous conditions such an inflammatory liver diseases and also in smokers. It is important that immunoassays used for the monitoring of cancer patient status not be interfered with by these other CEA-like antigens. Conversely, it is important to be able to distinguish the antigens by immunoassays because of the possibility that different tumor types may preferentially express different forms of CEA. If so, then the ability to reliably measure the different forms of CEA can provide the means to diagnose or more successfully treat different forms of cancer.
The members of the xe2x80x9cCEA familyxe2x80x9d share some antigenic determinants. These common epitopes are not useful in distinguishing the members of the antigen family and antibodies recognizing them are of little use for measuring tumor-specific CEA levels.
U.S. Pat. No. 3,663,684, entitled xe2x80x9cCarcinoembryonic Antigen and Diagnostic Method Using Radioactive Iodinexe2x80x9d, concerns purification and radioiodination of CEA for use in a RIA.
U.S. Pat. No. 3,697,638 describes that CEA is a mixture of antigens (components A and B in this case). U.S. Pat. No. 3,697,638 mentions methods for separating and radioiodinating each component and their use in specific RIA""s.
U.S. Pat. No. 3,852,415, entitled xe2x80x9cCompositions for Use in Radioimmunoassay, as Substitute for Blood Plasma Extract in Determination of Carcinoembryonic Antigenxe2x80x9d relates to the use of a buffer containing EDTA and bovine serum albumin as a substitute for plasma as a diluent for CEA RIA""s.
U.S. Pat. No. 3,867,363, entitled xe2x80x9cCarcinoembryonic Antigensxe2x80x9d, is directed to the isolation of CEA components A and B, their labelling and use in a RIA.
U.S. Pat. No. 3,927,193, entitled xe2x80x9cLocalization of Tumors by Radiolabelled Antibodiesxe2x80x9d, concerns the use of radiolabelled anti-CEA antibodies in whole body tumor imaging.
U.S. Pat. No. 3,956,258, entitled xe2x80x9cCarcinoembryonic Antigensxe2x80x9d, relates to the isolation of CEA components A and B.
U.S. Pat. No. 4,086,217, entitled xe2x80x9cCarcinoembryonic Antigensxe2x80x9d, is directed to the isolation of CEA components A and B.
U.S. Pat. No.4,140,753, entitled xe2x80x9cDiagnostic Method and Reagentxe2x80x9d, concerns the purification of a CEA isomer called CEA-S1 and its use in a RIA.
U.S. Pat. No. 4,145,336, entitled xe2x80x9cCarcinoembryonic Antigen Isomerxe2x80x9d, relates to the antigen CEA-S1.
U.S. Pat. No. 4,180,499, entitled xe2x80x9cCarcinoembryonic Antigensxe2x80x9d, describes a process for producing CEA component B.
U.S. Pat. No. 4,228,236, entitled xe2x80x9cProcess of Producing Carcinoembryonic Antigenxe2x80x9d, is directed to the use of the established cell lines LS-174T and LS-180 or clones or derivatives thereof for the production of CEA.
U.S. Pat. No. 4,272,504, entitled xe2x80x9cAntibody Adsorbed Support Method for Carcinoembryonic Antigen Assayxe2x80x9d, concerns two concepts for the radioimmunoassay of CEA. First, U.S. Pat. No. 4,272,504 relates to a sample pretreatment in the form of heating to 65 to 85xc2x0 C. at pH 5 to precipitate and eliminate extraneous protein. Second, it describes the use of a solid phase antibody (either on beads or tubes) as a means to capture analyte and radiolabelled CEA tracer.
U.S. Pat. No. 4,299,815, entitled xe2x80x9cCarcinoembryonic Antigen Determinationxe2x80x9d, concerns diluting a CEA sample with water and pretreating by heating to a temperature below which precipitation of protein will occur. The pretreated sample is then immunoassayed using RIA, EIA, FIA or chemiluminescent immunoassay.
U.S. Pat. No. 4,349,528, entitled xe2x80x9cMonoclonal Hybridoma Antibody Specific for High Molecular Weight Carcinoembryonic Antigenxe2x80x9d, is directed to a monoclonal antibody reacting with 180 kD CEA, but not with other molecular weight forms.
U.S. Pat. No. 4,467,031, entitled xe2x80x9cEnzyme-Immunoassay for Carcinoembryonic Antigenxe2x80x9d, relates to a sandwich enzyme immunoassay for CEA in which the first of two anti-CEA monoclonal antibodies is attached to a solid phase and the second monoclonal is conjugated with peroxidase.
U.S. Pat. No. 4,489,167, entitled xe2x80x9cMethods and Compositions for Cancer Detectionxe2x80x9d, describes that CEA shares an antigenic determinant with alpha-acid glycoprotein (AG), which is a normal component of human serum. The method described therein concerns a solid-phase sandwich enzyme immunoassay using as one antibody an antibody recognizing AG and another antibody recognizing CEA, but not AG.
U.S. Pat. No. 4,578,349, entitled xe2x80x9cImmunoassay for Carcinoembryonic Antigen (CEA)xe2x80x9d, is directed to the use of high salt containing buffers as diluents in CEA immunoassays.
EP 113072-A, entitled xe2x80x9cAssaying Blood Sample for Carcinoembryonic Antigenxe2x80x94After Removal of Interfering Materials by Incubation with Silica Gelxe2x80x9d, relates to the removal from a serum of a plasma sample of interfering substances by pretreatment with silica gel. The precleared sample is then subjected to an immunoassay.
EP 102008-A, entitled xe2x80x9cCancer Diagnostics Carcinoembryonic Antigenxe2x80x94Produced from Perchloric Acid Extracts Without Electrophoresisxe2x80x9d, relates to a procedure for the preparation of CEA from perchloric acid extracts, without the use of an electrophoresis step.
EP 92223-A, entitled xe2x80x9cDetermination of Carcinoembryonic Antigen in Cytosol or Tissuexe2x80x94for Therapy Control and Early Recognition of Regressionxe2x80x9d, concerns an immunoassay of CEA, not in serum or plasma, but in the cytosol fraction of the tumor tissue itself.
EP 83103759.6, entitled xe2x80x9cCytosole-CEA-Measurement as Predictive Test in Carcinoma, Particularly Mammacarcinomaxe2x80x9d, is similar to EP 92223-A.
EP 83303759, entitled xe2x80x9cMonoclonal Antibodies Specific to Carcinoembryonic Antigenxe2x80x9d, relates to the production of xe2x80x9cCEA specificxe2x80x9d monoclonal antibodies and their use in immunoassays.
WO 84/02983, entitled xe2x80x9cSpecific CEA-Family Antigens, Antibodies Specific Thereto and Their Methods of Usexe2x80x9d, is directed to the use of monoclonal antibodies to CEA-meconium (MA)-, and NCA-specific epitopes in immunoassays designed to selectively measure each of these individual components in a sample.
All of the heretofore CEA assays utilize either monoclonal or polyclonal antibodies which are generated by immunizing animals with the intact antigen of choice. None of them address the idea of making sequence specific antibodies for the detection of a unique primary sequence of the various antigens. They do not cover the use of any primary amino acid sequence for the production of antibodies to synthetic peptides or fragments of the natural product. They do not include the concept of using primary amino acid sequences to distinguish the CEA family members. None of them covers the use of DNA or RNA clones for isolating the genes with which to determine the primary sequence.
Nucleotidexe2x80x94A monomeric unit of DNA or RNA containing a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon (1xe2x80x2 carbon of the pentose) and that combination of base and sugar is called a nucleoside. The base characterizes the nucleotide. The four DNA bases are adenine (xe2x80x9cAxe2x80x9d), guanine (xe2x80x9cGxe2x80x9d), cytosine (xe2x80x9cCxe2x80x9d), and thymine (xe2x80x9cTxe2x80x9d). The four RNA bases are A, G, C and uracil (xe2x80x9cUxe2x80x9d).
DNA Sequencexe2x80x94A linear array of nucleotides connected one to the other by phosphodiester bonds between the 3xe2x80x2 and 5xe2x80x2 carbons of adjacent pentoses.
Functional equivalentsxe2x80x94It is well known in the art that in a DNA sequence some nucleotides can be replaced without having an influence on the sequence of the expression product. With respect to the peptide this term means that one or more amino acids which have no function in a particular use can be deleted or replaced by another one.
Codonxe2x80x94A DNA sequence of three nucleotides (a triplet) which encodes through mRNA an amino acid, a translation start signal or a translation termination signal. For example, the nucleotide triplets TTA, TTG, CTT, CTC, CTA and CTG encode the amino acid leucine (xe2x80x9cLeuxe2x80x9d), TAG, TAA and TGA are translation stop signals and ATG is a translation start signal.
Reading Framexe2x80x94The grouping of codons during translation of mRNA into amino acid sequences. During translation, the proper reading frame must be maintained. For example, the sequence GCTGGTTGTAAG may be translated in three reading frames or phases, each of which affords a different amino acid sequence
GCT GGT TGT AAGxe2x80x94Ala-Gly-Cys-Lys
G CTG GTT GTA AGxe2x80x94Leu-Val-Val
GC TGG TTG TAA Gxe2x80x94Trp-Leu-(STOP).
Polypeptidexe2x80x94A linear array of amino acids connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent amino acids.
Genomexe2x80x94The entire DNA of a cell or a virus. It includes inter alia the structural genes coding for the polypeptides of the cell or virus, as well as its operator, promoter and ribosome binding and interaction sequences, including sequences such as the Shine-Delgarno sequences.
Structural Genexe2x80x94A DNA sequence which encodes through its template or messenger RNA (xe2x80x9cmRNAxe2x80x9d) a sequence of amino acids characteristics of a specific polypeptide.
Transcriptionxe2x80x94The process of producing mRNA from a structural gene.
Translationxe2x80x94The process of producing a polypeptide from mRNA.
Expressionxe2x80x94The process undergone by a structural gene to produce a polypeptide. It is a combination of transcription and translation.
Plasmidxe2x80x94A non-chromosomal double-stranded DNA sequence comprising an intact xe2x80x9crepliconxe2x80x9d such that the plasmid is replicated in a host cell. When the plasmid is placed within a unicellular organism, the characteristics of that organism may be changed or transformed as a result of the DNA of the plasmid. For example, a plasmid carrying the gene for tetracycline resistance (TetR) transforms a cell previously sensitive to tetracycline into one which is resistant to it. A cell transformed by a plasmid is called a xe2x80x9ctransformantxe2x80x9d.
Phage or Bacteriophagexe2x80x94Bacterial virus, many of which consist of DNA sequences encapsulated in a protein envelope or coat (xe2x80x9ccapsid proteinxe2x80x9d).
Cloning Vehiclexe2x80x94A plasmid, phage DNA or other DNA sequence which is capable of replicating in a host cell, which is characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without attendant loss of an essential biological function of the DNA, e.g., replication, production of coat proteins or loss of promoter or binding sites, and which contains a marker suitable for use in the identification of transformed cells, e.g., tetracycline resistance or ampicillin resistance. A cloning vehicle is often called a vector.
Cloningxe2x80x94The process of obtaining a population of organisms or DNA sequences derived from one such organism or sequence by asexual reproduction.
Recombinant DNA Molecule or Hybrid DNAxe2x80x94A molecule consisting of segments of DNA from different genomes which have been joined end-to-end outside of living cells and have the capacity to infect some host cell and be maintained therein.
cDNA Expression Vectorxe2x80x94A procaroytic cloning vehicle which also contains sequences of nucleotides that facilitate expression of cDNA sequences in eucaroytic cells. These nucleotides include sequences that function as eucaryotic promoter, alternative splice sites and polyadenylation signals.
Transformation/Transfectionxe2x80x94DNA or RNA is introduced into cells in such a way as to allow gene expression. xe2x80x9cInfectedxe2x80x9d referred to herein concerns the introduction of RNA or DNA by a viral vector into the host.
xe2x80x9cInjectedxe2x80x9d referred to herein concerns the microinjection (use of a small syringe) of DNA into a cell.
CEA antigen family (CEA gene family)xe2x80x94a set of genes (gene family) and their products (antigen family) that share nucleotide sequences homologous to partial cDNA LV-7 (CEA -(a)) and as a result of theses similarities also share a subset of their antigenic epitopes. Examples of the CEA antigen family include CEA (=CEA-(b)), transmembrane CEA (TMCEA)=(CEA-(c) and normal crossrecting antigen NCA (=CEA-(d)).
The present invention concerns the following DNA sequences designed as TM-2 (CEA-(e)), TM-3 (CEA-(f)), TM-4 (CEA-(g)), KGCEA1 and KGCEA2, which code for CEA antigen family peptide sequences or nucleic acids having a base sequence (DNA or RNA) that are hybridizable therewith:
The present invention is also directed to a replicable recombinant cloning vehicle (xe2x80x9cvectorxe2x80x9d) having an insert comprising a nucleic acid, e.g., DNA, which comprises a base sequence which codes for a CEA peptide or a base sequence hybridizable therewith.
This invention also relates to a cell that is transformed/transfected, infected or injected with the above described replicable recombinant cloning vehicle or nucleic acid hybridizable with the aforementioned cDNA. Thus the invention also concerns the transfection of cells using free nucleic acid, without the use of a cloning vehicle.
Still further, the present invention concerns a polypeptide expressed by the above described transfected, infected or injected cell, which polypeptide exhibits immunological cross-reactivity with a CEA, as well as labelled forms of the polypeptide. The invention also relates to polypeptides having an amino acid sequence, i.e., synthetic peptides, or the expression product of a cell that is transfected, injected, infected with the above described replicable recombinant cloning vehicles, as well as labelled forms thereof. Stated otherwise, the present invention concerns a synthetic peptide having an amino acid sequence corresponding to the entire amino acid sequence or a portion thereof having no less than five amino acids of the aforesaid expression product.
The invention further relates to an antibody preparation specific for the above described polypeptide.
Another aspect of the invention concerns an immunoassay method for detecting CEA or a functional equivalent thereof in a test sample comprising
(a) contacting the sample with the above described antibody preparation, and
(b) determining binding thereof to CEA in the sample.
The invention also is directed to a nucleic acid hybridization method for detecting a CEA or a related nucleic acid (DNA or RNA) sample in a test sample comprising
(a) contacting the test sample with a nucleic acid probe comprising a nucleic acid, which comprises a base sequence which codes for a CEA peptide sequence or a base sequence that is hybridizable therewith, and
(b) determining the formation of the resultant hybridized probe.
The present invention also concerns a method for detecting the presence of carcinoembryonic antigen or a functional equivalent thereof in an animal or human patient in vivo comprising
a) introducing into said patient a labeled (e.g., a radio-opaque material that can be detected by X-rays, radiolabeled or labeled with paramagnetic materials that can be detected by NMR) antibody preparation according to the present invention and
b) detecting the presence of such antibody preparation in the patient by detecting the label.
In another aspect, the present invention relates to the use of an antibody preparation according to the present invention for therapeutic purposes, namely, attaching to an antibody preparation radionuclides, toxins or other biological effectors to form a complex and introducing an effective amount of such complex into an animal or human patient, e.g., by injection or orally. The antibody complex would attach to CEA in a patient and the radionuclide, toxin or other biological effector would serve to destroy the CEA expressing cell.